The use of messenger RNA (mRNA) in gene therapy is increasing in recent years, due to its unique features compared to plasmid DNA: Transient expression, no need to enter into the nucleus and no risk of insertional mutagenesis. Nevertheless, the clinical application of mRNA as a therapeutic tool is limited by its instability and ability to activate immune responses; hence, mRNA chemical modifications together with the design of suitable vehicles result essential. This manuscript includes a revision of the strategies employed to enhance in vitro transcribed (IVT) mRNA functionality and efficacy, including the optimization of its stability and translational efficiency, as well as the regulation of its immunostimulatory properties. An overview of the nanosystems designed to protect the mRNA and to overcome the intra and extracellular barriers for successful delivery is also included. Finally, the present and future applications of mRNA nanomedicines for immunization against infectious diseases and cancer, protein replacement, gene editing, and regenerative medicine are highlighted.

The development of safe and effective nucleic acid delivery systems remains a challenge, with solid lipid nanoparticle (SLN)-based vectors as one of the most studied systems. In this work, different SLNs were developed, by combination of cationic and ionizable lipids, for delivery of mRNA and pDNA. The influence of formulation factors on transfection efficacy, protein expression and intracellular disposition of the nucleic acid was evaluated in human retinal pigment epithelial cells (ARPE-19) and human embryonic kidney cells (HEK-293). A long-term stability study of the vectors was also performed. The mRNA formulations induced a higher percentage of transfected cells than those containing pDNA, mainly in ARPE-19 cells; however, the pDNA formulations induced a greater protein production per cell in this cell line. Protein production was conditioned by energy-dependent or independent entry mechanisms, depending on the cell line, SLN composition and kind of nucleic acid delivered. Vectors containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as unique cationic lipid showed better stability after seven months, which improved with the addition of a polysaccharide to the vectors. Transfection efficacy and long-term stability of mRNA vectors were more influenced by formulation-related factors than those containing pDNA; in particular, the SLNs containing only DOTAP were the most promising formulations for nucleic acid delivery.

One of the main challenges in gene therapy is the issue of delivery, and it is especially relevant for the success of gene therapy in the cornea. In the present work, eye drops containing biocompatible non-viral vectors based on solid lipid nanoparticles (SLNs) as gene delivery systems to induce the expression of interleukin 10 (IL-10) were designed to address the treatment of corneal inflammation. Two kinds of SLNs combined with different ligands (protamine, dextran, or hyaluronic acid (HA)) and formulated with polyvinyl alcohol (PVA) were prepared. SLN-based vectors were characterized in terms of size, adhesiveness, viscosity, and pH, before topical administration to wild type and IL-10 knock out (KO) mice. The formulations showed a homogenous particle size below 400 nm and a positive surface charge to favor bioadhesion; the incorporation of PVA improved the corneal penetration. After three days of treatment by topical instillation, SLN-based vectors mainly transfected corneal epithelial cells, HA-formulations being the most effective ones. IL-10 was capable of reaching even the endothelial layer. Corneal sections showed no histological change and formulations seemed to be well tolerated after repeated topical administration. These promising results highlight the possible contribution of non-viral gene augmentation therapy to the future clinical approach of corneal gene therapy.

Gene silencing targeting proangiogenic factors have been shown to be a useful strategy in the treatment of corneal neovascularization (CNV). Among interference RNA (RNAi) molecules, short-hairpin RNA (shRNA) is a plasmid-coded RNA able to down-regulate the expression of the desired gene. It is continuously produced in the host cell, inducing a durable gene silencing effect. The aim of this work was to develop a solid lipid nanoparticle (SLN)-based shRNA delivery system to downregulate metalloproteinase 9 (MMP-9), a proangiogenic factor, in corneal cells for the treatment of CNV associated with inflammation. The nanovectors were prepared using a solvent emulsification-evaporation technique, and after physicochemical evaluation, they were evaluated in different culture cell models. Transfection efficacy, cell internalization, cell viability, the effect on MMP-9 expression, and cell migration were evaluated in human corneal epithelial cells (HCE-2). The inhibition of tube formation using human umbilical vein endothelial cells (HUVEC) was also assayed. The non-viral vectors based on SLN were able to downregulate the MMP-9 expression in HCE-2 cells via gene silencing, and, consequently, to inhibit cell migration and tube formation. These results demonstrate the potential of lipid nanoparticles as gene delivery systems for the treatment of CNV-associated inflammation by RNAi technology.

The anti-inflammatory cytokine Interleukin-10 (IL-10) is considered an efficient treatment for corneal inflammation, in spite of its short half-life and poor eye bioavailability. In the present work, mRNA-based nanomedicinal products based on solid lipid nanoparticles (SLNs) were developed in order to produce IL-10 to treat corneal inflammation. mRNA encoding green fluorescent protein (GFP) or human IL-10 was complexed with different SLNs and ligands. After, physicochemical characterization, transfection efficacy, intracellular disposition, cellular uptake and IL-10 expression of the nanosystems were evaluated in vitro in human corneal epithelial (HCE-2) cells. Energy-dependent mechanisms favoured HCE-2 transfection, whereas protein production was influenced by energy-independent uptake mechanisms. Nanovectors with a mean particle size between 94 and 348 nm and a positive superficial charge were formulated as eye drops containing 1% (w/v) of polyvinyl alcohol (PVA) with 7.1–7.5 pH. After three days of topical administration to mice, all formulations produced GFP in the corneal epithelium of mice. SLNs allowed the obtaining of a higher transfection efficiency than naked mRNA. All formulations produce IL-10, and the interleukin was even observed in the deeper layers of the epithelium of mice depending on the formulation. This work shows the potential application of mRNA-SLN-based nanosystems to address corneal inflammation by gene augmentation therapy.

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.